7. Project Summary/Abstract. Staphylococcus aureus colonizes 30-50% of the population and is the leading cause of skin and soft tissue infections, osteomyelitis, pneumonia and endocarditis. At a time when hospital-acquired staphylococcal disease is decreasing, particularly dangerous clones of S. aureus pose a major health threat to otherwise healthy individuals, especially children. These community-acquired (CA) infections are the most frequent cause of emergency room visits and are resistant to a number of first- and last-line antibiotics (for example, amoxicillin and vancomycin). The use and misuse of these antibiotics promotes the rise of resistance by selecting for growth; therefore it is preferable to disable the ability to cause disease than to kill the bacteria. Previous work has shown that the S. aureus regulatory protein CodY helps to suppress the production of virulence factors when activated by the amino acids isoleucine, leucine and valine (ILV), and guanosine triphosphate (GTP). In the absence of active CodY and suppression, toxins and other disease-causing factors are elevated and those bacteria are significantly more virulent. Moreover, maintaining activity during growth keeps CodY-dependent genes repressed. Therefore, CodY could be exploited as an anti-virulence therapy and allow at-risk patients to clear the infection naturally. Before doing so, it is essential to establish that CodY activity changes within tissues during infection. If so, administering analogs of ILV and guanine nucleotides that prevent loss of CodY activity could potentially prevent staphylococcal disease. In this proposal we seek to do two things to reveal the relationship between CodY, host nutrients, and regulation of gene expression: i.) assess whether host tissue affects CodY-dependent gene expression using cutting edge in vivo and ex vivo live imaging and fluorescent reporters, and ii.) determine whether increasing ILV abundance in staphylococci during infection affects pathogenicity. We will collaborate with CA-staphylococcal biologist Michael Otto (National Institutes of Health), and preclinical imaging experts led by Christopher Albanese (Georgetown University Medical Center). Together, we will employ a multifaceted approach to gain a deeper understanding of CodY function in vivo and to test this anti-virulence approach.